Decreased 1,25-Dihydroxyvitamin D3 level is involved in the pathogenesis of Vogt-Koyanagi-Harada (VKH) disease.

Purpose 1,25-Dihydroxyvitamin D3 [1,25(OH)2D3] has recently been found to be involved in the development of autoimmune diseases. This study was to investigate the expression and potential role of 1,25(OH)2D3 in the pathogenesis of Vogt-Koyanagi-Harada (VKH) disease. Methods Blood samples were obtained from VKH patients and healthy individuals. Serum 1,25(OH)2D3 levels were measured using ELISA. Peripheral blood mononuclear cells (PBMCs) or cluster of differentiation (CD) 4+ T cells were cultured with or without 1,25(OH)2D3 in the presence of anti-CD3 and anti-CD28 for the measurement of cell proliferation and cytokines. The cell proliferation was detected using the Cell Counting Kit. The levels of interleukin (IL)-17 and interferon (IFN)-γ levels in the supernatants of PBMCs or CD4+ T cells were detected by ELISA. Results 1,25(OH)2D3 was significantly decreased in the serum of active VKH patients as compared with inactive VKH patients and controls. It significantly inhibited PBMCs proliferation and CD4+ T cell proliferation. It was also able to significantly inhibit the production of IL-17 and IFN-γ by both PBMCs and CD4+ T cells from VKH patients and controls. Conclusions These findings suggest that decreased expression of 1,25(OH)2D3 may be involved in the development of VKH disease. 1,25(OH)2D3 may be potentially used in the treatment of this disease.

Vitamin D is produced in the skin upon exposure to sunlight or obtained from the diet [1][2][3]. Its receptor has been found in the immune cells and parts of these cells are able to produce Vitamin D3 [4][5][6][7]. 1,25(OH)2D3, the biologically active metabolite of Vitamin D3, has recently been shown to have immunomodulation property beside its involvement in the bone and calcium metabolism [8,9]. Recent studies showed that 1,25(OH)2D3 was able to skew the T cell compartment into a more anti-inflammatory and regulated state, as evidenced by inhibition of Th1 and Th17 cells and promotion of Th2 and T reg cells [10,11]. In Vitro experiments showed that 1,25(OH)2D3 could promote both innate and adaptive immune responses. 1,25(OH)2D3 receptor gene knockout mice showed a significantly higher susceptibility to several autoimmune disease models, such as experimental autoimmune encephalomyelitis [12,13], experimental autoimmune uveitis [14], and experimental allergic asthma [15]. Additionally, serum level of 1,25(OH)2D3 was found to be decreased in several human autoimmune diseases, such as multiple sclerosis [16][17][18], rheumatoid arthritis [19,20], Behçet's disease [21], Graves disease [22], and systemic lupus erythematosus [23,24]. All these results suggest that 1,25(OH)2D3 is a negative factor and that down-regulated expression of 1,25(OH)2D3 is possibly involved in the pathogenesis of these diseases.  [25][26][27]. It is one of the most common uveitis entities in China as well as in the Far East of Asia [26,[28][29][30]. Studies have showed that both enhanced T helper (Th) 1 and Th17 cell responses are involved in the pathogenesis of this disease.
In this study we examined the expression of 1,25(OH)2D3 and its possible involvement in the increased Th1 and Th17 response as already reported previously. Our results showed that a down-regulated expression of 1,25(OH)2D3 was present in active VKH patients and was possibly one of factors responsible for the increased Th1 and Th17 response in this disease.

Patients and controls:
A total 25 adult patients with VKH disease (13 men and 12 women) with an average age of 38.4 years and a total 16 healthy individuals (9 men and 7 women) with an average age of 40.5 years were included in the study. All patients were diagnosed as having definite VKH disease according to the diagnostic criteria revised for VKH disease in an international committee on nomenclature [31]. Fifteen patients showed active intraocular inflammation (active uveitis stage), as evidenced by mutton fat keratic precipitates, cells in the anterior chamber and sunset glow fundus. The systemic findings included headache (54%), poliosis and alopecia (72%), vitiligo (23%), and tinnitus (46%). Ten active patients were at their first attack with disease duration <6 months, and 5 active patients showed recurrent intraocular inflammation with the duration of <2 years. Ten inactive VKH patients showed typical bilateral sunset glow fundus but without any intraocular inflammation (inactive uveitis stage), with the disease duration from 1 to 3 years. No systemic corticosteroids or other immunosuppressive agents were used at least for 2 weeks before being referred to our hospital and blood sampling in active VKH patients. Ten patients without active intraocular inflammation for at least 3 months after the treatment of corticosteroid and immunosuppressant were chosen as controls. No patients had received any dietary supplements before study entry. Blood samples were obtained from these active or inactive patients during November 1, 2009 to March 31, 2010, and the daily sun exposure time was estimated to be <1.5 h for all subjects. This study was approved by the Ethics Committee of the First Affiliated Hospital of Chongqing Medical University, and complied with the tenets of the Declaration of Helsinki.

1,25(OH)2D3 assays:
Blood samples were collected from 8 active VKH patients, 7 inactive VKH patients and 8 healthy controls. Serum was obtained by centrifugation at 3,000× g for 10 min and stored at −70 °C until analysis. Serum 1,25(OH)2D3 level was measured using a commercially available ELISA kit (Immunodiagnostik, Bensheim, Germany) according to manufacturer's instructions. An assay sensitivity level was 6.0 pg/ml.

Effect of 1,25(OH)2D3 on proliferation of PBMCs and CD4 + T cells:
A primary experiment with different concentrations of 1,25(OH)2D3 showed that it significantly inhibited the proliferation of PBMCs from active VKH patients and controls. Furthermore, the result showed that its inhibitory effect was in a dose-dependent manner ( Figure 2B). As 1×10 −7 mol/l of 1,25(OH)2D3 could induce a significant suppression on the proliferation of PBMCs, this concentration was used in the following experiments. Both PBMCs and CD4 + T cells from active VKH patients showed a significantly higher cell proliferation as compared with the inactive VKH patients and controls. 1,25(OH)2D3 significantly inhibited the cell proliferation of PBMCs and CD4 + T cells from all VKH patients and controls ( Figure 2C). There was no difference among these tested three groups concerning the inhibitory percentage.

Effect of 1,25(OH)2D3 on the production of IL-17:
PBMCs and CD4 + T cells from VKH patients and controls cultured with 1,25(OH)2D3 in the presence of anti-CD3 and anti-CD28 antibodies were used to evaluate the influence of this molecule on IL-17 production. A significantly increased production of IL-17 was observed in active VKH patients as compared with inactive VKH patients (PBMCs: p=0.039; CD4 + T cells: p=0.015) and controls (PBMCs: p=0.001; CD4 + T cells: p=0.002). 1,25(OH)2D3 significantly inhibited the production of IL-17 by PBMCs and CD4 + T cells from the VKH patients and controls ( Figure 3A,B). There was no difference among the tested three groups concerning the inhibitory percentage.

Effect of 1,25(OH)2D3 on the production of IFN-γ: PBMCs and CD4 + T cells from VKH patients and controls cultured with 1,25(OH)2D3 in the presence of anti-CD3 and anti-CD28
antibodies were used to examine the influence of this molecule on the production of IFN-γ. IFN-γ production by both PBMCs and CD4 + T was significantly higher in active VKH patients as compared with inactive VKH patients (PBMCs: p=0.001, CD4 + T cells: p=0.018) and normal controls (PBMCs: p=0.008, CD4 + T cells: p=0.005). 1,25(OH)2D3 significantly down-regulated the production of IFN-γ by PBMCs and CD4 + T cells from VKH patients and controls ( Figure 4A,B). Similar to the IL-17 result, no difference was found concerning the inhibitory percentage of 1,25(OH)2D3 on the production of IFN-γ by PBMCs and CD4 + T cells among the tested three groups.

DISCUSSION
In this study, we showed that serum level of 1,25(OH)2D3 was significantly decreased in the serum of active VKH patients. 1,25(OH)2D3 was able to inhibit cell proliferation of PBMCs and CD4 + T cells. It also down-regulated the IL-17 and IFNγ production by activated PBMCs and CD4 + T cells from both VKH patients and controls. These results suggested that the decreased expression of 1,25(OH)2D3 may be involved in the development of VKH disease.
Vitamin D is a member of the steroid thyroid superfamily of nuclear receptors. The immune regulatory effects observed in other autoimmune diseases stimulated us to investigate whether 1,25(OH)2D3 was involved in the pathogenesis of VKH disease. For this purpose, we first detected the 1,25(OH)2D3 level in the serum of VKH patients. Our results showed that the serum level of 1,25(OH)2D3 was significantly decreased in active VKH patients. This result is generally consistent with those reported in other autoimmune diseases, such as multiple sclerosis [16,17], rheumatoid arthritis [19], and systemic lupus erythematosus [23,24]. This result suggest that downregulated expression of 1,25(OH)2D3 was associated with the active intraocular inflammation in VKH patients.
The decreased expression of 1,25(OH)2D3 in active VKH diseases, but not in inactive VKH patients, suggests that it may be involved in the pathogenesis of the intraocular inflammation in this disease. As T cell proliferation is one of important factors in the immune response or in the inflammation, we further investigated the effect of 1,25(OH)2D3 on cell proliferation. Our results showed that 1,25(OH)2D3 significantly inhibited the cell proliferation of PBMCs and CD4 + T cells from all VKH patients and controls. These results suggest that PBMCs and CD4 + T cells from VKH patients and controls are similar in the sensitivity to it with respect to cell proliferation. Our result is consistent with early report, in which 1,25(OH)2D3 was found to suppress cell proliferation of PBMCs or CD4 + cells in both human and animal models [19,24,33].
As IL-17 is found to have an important role in autoimmune diseases including VKH disease, we further examined the effect of 1,25(OH)2D3 on the secretion of IL-17. Consistent with our previous study [34], a significantly increased production of IL-17 was observed in active VKH patients. 1,25(OH)2D3 significantly inhibited the production of IL-17 by PBMCs and CD4 + T cells from VKH patients and controls. These results demonstrated an inhibitory effect of 1,25(OH)2D3 on IL-17 production reported in other autoimmune diseases [19,24,33]. Unexpectedly, we failed to find any difference regarding the inhibitory percentage of  1,25(OH)2D3 on the IL-17 production among the tested three groups, suggesting that the sensitivity of PBMCs and CD4 + T cells to 1,25(OH)2D3 is not different concerning IL-17 production.
Upregulated Th1 response has been found as one of the mechanisms of autoimmune diseases including VKH disease [34][35][36]. Our study further examined whether 1,25(OH)2D3 could influence the expression of IFN-γ, a typical cytokine of Th1 cells, in VKH patients. The result showed a significantly increased IFN-γ production by PBMCs and CD4 + T cells in active VKH patients. This result is consistent with the results reported previously [34]. Our study also showed that 1,25(OH)2D3 was able to inhibit the production of IFN-γ by PBMCs and CD4 + T cells from both VKH patients and controls. Therefore, the sensitivity of both PBMCs and CD4 + T cell to 1,25(OH)2D3 were not different among the tested three groups concerning IFN-γ production.
In summary, our study revealed that decreased 1,25(OH)2D3 level was associated with active intraocular inflammation in VKH patients. 1,25(OH)2D3 could significantly inhibit cell proliferation of PBMCs and CD4 + T cells, and down-regulated expression of IL-17 and IFN-γ.
These results suggest that down-regulated expression of 1,25(OH)2D3 may be one of mechanisms for the increased IL-17 and IFN-γ in the development of VKH disease. Our study also suggests that upregulating 1,25(OH)2D3 might provide a new strategy for the treatment of VKH disease.  anti-CD3 and anti-CD28 antibodies in the presence or absence of 1,25(OH)2D3 for 72 h. A: IFN-γ production by PBMCs from active VHK patients (n=11), inactive VHK patients (n=7) and normal controls (n=12). B: IFN-γ production by CD4+ T cells from active VHK patients (n=7), inactive VHK patients (n=7) and normal controls (n=9).